Injection of cells is currently only a viable technique in a limited number of fields, for example in vitro fertilisation, and currently is carried out manually and individually on each cell. It requires a high level of skill and an experienced operator can only inject in the order of one cell per minute. There are many other fields that would benefit from cell injection of macromolecules, genes, chromosomes, organelles, or any other substance desired to be injected into a cell were it possible to achieve this on a large numbers of cells. Gene therapy, biotechnology, life sciences research, diagnostics, pharmaceutical and agrochemical research are among many fields that would benefit from a more facile high throughput cell injection method.
Currently using manual techniques the cells are suspended in solution and each cell is individually injected by fixing a cell into position by the operator “sucking” the cell onto the end of a narrow pipette. Whilst watching the operation through a microscope the operator then inserts a needle into the cell. Once the injection is made the needle is retracted manually and the cell released, then the next cell is injected and so on. In addition variations of this basic manual technique are available such as, for example, for injecting cells which are attached to a dish as a monolayer. The cost of injecting a small number of cells is expensive and means that microinjection of cells is not a technique used widely in the pharmaceutical or agrochemical research.
We have devised of a device in which a large numbers of cells (hundreds, thousands or millions) may be micro-injected with minimal operator involvement by use of a microfabricated device which impels cells onto an injection needle.
Microfabrication techniques are generally known in the art using tools developed by the semiconductor industry to miniaturise electronics and it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use, for example, in simple analytical tests. See, e.g., Ramsey, J. M. et al. (1995), “Microfabricated chemical measurement Systems,” Nature Medicine 1:1093-1096; and Harrison, D. J. et al (1993), “Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip,” Science 261:895-897.
Devices made by micromachining planar substrates have been made and used for chemical separation, analysis, and sensing. See, e.g., Manz, A. et al. (1994), “Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis system,” J. Micromech. Microeng. 4:257-265.
We have been able to construct a microfabricated needle onto which cells may be impelled towards so that the needle pierces the cell and material may be injected or extracted from the cell.
We present as a first feature of the invention a microfabricated cell injector comprising an injection wall and projecting from the injection wall a cell injection needle, such that in use cells suspended in a fluid are impelled towards the injection wall and pierced by the injection needle whereupon material is (1) injected into the cell, (2) extracted from the cell, or (3) injected into the cell and then extracted from the cell the steps being in any order and any number of times.
We have also found that by careful arrangement of channels (microfluidic channels) formed within a microfabricated device a conduit is formed through which the flow of cells in a suspension may be controlled to an extent that cells may be injected by impelling them onto an injection needle individually.
We disclose as a further feature of the invention a microfabricated cell injector comprising an internal surface defining a conduit, which in use transports cells suspended in a fluid, the conduit having an inlet and an outlet, the conduit further comprising a cell injection needle, such that, in use cells enter the injector via the inlet, are moved along the conduit and are pierced by the cell injection needle whereupon material is (1) injected into the cell, (2) extracted from the cell, or (3) injected into the cell and then extracted from the cell the steps being in any order and any number of times, and the cells are then, optionally, moved to the outlet.
A further feature of the invention is a method for the microinjection of cells which method comprises passing a suspension of cells in a fluid through a conduit comprising a cell injection needle, the cells thereby being pierced by the injection needle and material is: (1) injected into the cell (2) extracted from the cell or (3) injected into the cell and then extracted from the cell the steps being in any order and any number of times; as the cells pass through the conduit.
It should be understood that the arrangement, type and dimensions of the device and the components therein will vary according to the use or application, as will become apparent. It is generally preferred that the microfabricated conduit only allows a single cell to be impelled upon a single injection needle at any one time.
In this disclosure, the term “microfabricated” includes, for example, devices capable of being fabricated on glass, plastic, silicon or any other suitable material. Suitable microfabrication techniques are those readily available to those practising the art of microfabrication and include such methods as LIGA, thermoplastic micropattern transfer, resin based microcasting, micromolding in capillaries (MIMIC), wet isotropic and anisotropic etching, laser assisted chemical etching (LACE), and reactive ion etching (RIE), or other techniques known within the art of microfabrication. In the case of silicon microfabrication, larger wafers will accommodate a plurality of the devices of this invention in a plurality of configurations. A few standard wafer sizes are 3″(7.5 cm), 4″(10 cm), 6″(15 cm), and 8″(20 cm). Application of the principles presented herein using new and emerging microfabrication methods or materials is within the scope and intent of the invention.
The injection needle has a diameter of dimensions comparable with the dimension of the cells to be injected, for example between 1% and 50% of the cell diameter, preferably between 5% and 30%. Typical cell diameters are from 10 microns to 50 microns, but will vary according to the cell origin and type. The walls of the injection needle when hollow may be from 1 micron thick and may be as narrow as 0.1 micron thick at the point of the needle. Where the injection needle has a hollow tip this is connected to a microfluidic channel which is able to deliver to the injection needle tip the material for injection. Preferably the injection needle is fixed in the device relative to the walls of the microfluidic channels which form the conduit such that it projects and injection is achieved by moving the cells on and off the injection needle, rather than by moving the injection needle into and out of the cell. Preferably the injection needle is positioned on a surface of the microfluidic channel, which we term the “injection wall”, see for example FIG. 1. The injection wall forms part of the “housing” for the needle which may be simply an integral part of the conduit or may be a distinct aspect which forms a suitable receptacle for receiving/positioning/holding the cell prior to injection. The length and shape of the injection needle that is exposed above the injection wall will determine the “injection depth”, that is the depth to which the injection needle will penetrate the cell. This depth will depend on the cell type, the design of the needle, and the application. In particular, it will depend on the cellular compartment that it is desired to inject into. For example, for injection into the cytoplasm, the injection depth could be in the order of 1 micron, for example 1 to 5 microns; whereas, for an injection into the nucleus, the injection depth will need to be greater, for example 3-10 microns. Given a knowledge of the cell type, it will be possible for the skilled bioscientist to select a device with the appropriate injection depth.
Preferably the needle is hollow and substantially circular in cross section, the external diameter of the needle continuously decreasing as it projects from the base of the needle to its tip, the tip being less than 25 microns, preferably less than 5 microns, in diameter. As a feature of the invention we present use of the above described needle in the piercing and injecting of material into, or extracting material from, cells.
The injection wall surrounding the immediate area of the injection needle may be permeable to the medium in which the cells are contained, but impermeable to the passing of cells. In certain orientations of the device permeable walls are preferred and allow the passing of the cell medium through the injection wall, facilitating the movement of the cell towards the injection needle. The permeability of the wall may be achieved by one or a small number of orifices positioned around the needle, preferably in a symmetrical fashion. It will be clear that a large number of designs could in principle achieve the aim of forcing the cell onto the needle and these are incorporated into the invention. The injection wall may optionally itself be charged to attract the cell towards the injection needle, or reverse charged to expel the cell from the injection needle. Alternatively the charge may alternate to impel the cell onto the needle and then to expel it. The injection wall may be flat or any other shape to accommodate the cell whilst it is on the injection needle.